The prevalence of type 2 diabetes in people over 60 years of age approaches 25%, yet the pathogenesis of diabetes in this group remains poorly understood. Insulin resistance in skeletal muscle is a major factor in the development of type 2 diabetes and recent studies have demonstrated a strong relationship between accumulation of intramyocellular lipid (IMCL) content and defects in insulin stimulated muscle glycogen synthesis due to decreased glucose transport activity. Whether this same mechanism is responsible for insulin resistance in the elderly is unknown. In this proposal we plan to use state-of-the-art NMR techniques combined with GC/MS and LC/MS/MS techniques to explore the mechanism of insulin resistance in the elderly. Specifically, 13C NMR spectroscopy will be used to examine rates of insulin stimulated muscle glycogen synthesis in healthy lean non-smoking older (60-75 yrs) subjects compared to healthy, younger (18-30 yrs) non-smoking subjects matched for gender, body weight, percent fat mass and physical activity. Rate controlling steps in this process (i.e. glucose transport, hexokinase, glycogen synthase) will be examined using 31p and 13C NMR spectroscopy to measure intracellular concentrations of glucose-6- phosphate and glucose. Localized IMCL content in skeletal muscle will be assessed with 1H NMR spectroscopy. Since preliminary data suggest that IMCL content is increased in the elderly we also plan to examine whether this increase in IMCL content is due to increased delivery of fatty acids to skeletal muscle, by employing microdialysis measurements of glycerol release from peripheral fat, to assess localized rates of lipolysis, along with [2H] glycerol turnover measurements to assess rates of whole body lipolysis. Finally, to determine whether these age related increases in IMCL content might be attributed to decreased mitochondrial energy metabolism, we will employ a novel 31P/13C NMR method to assess rates of mitochondrial ATP synthesis and tricarboxylic acid cycle flux in skeletal muscle. Overall, it is anticipated that the results from these studies will provide new insights into the pathogenesis of insulin resistance in the elderly, which in turn will lead to potentially new therapeutic targets to prevent or reverse diabetes in these individuals.